Chemically activating cAMP Production in M. tuberculosis blocks bacterial cholesterol metabolism and shows therapeutic potential

Kaley M. Wilburn1, Christine R. Montague1, Ashley K. Woods2, Bo Qin2, Theresa L. Southard3, Michael Petrassi2, Brian C. VanderVen1

1 Microbiology & Immunology, Cornell University, Ithaca, NY 14853 USA.

2 Calibr at Scripps Research, La Jolla, CA 92037, USA.

3 Biomedical Sciences, Cornell University, Ithaca, NY 14853 USA.

Identifying new drug candidates with novel mechanisms of action that simplify or shorten current TB treatments would address a major unmet need in TB control measures. It is generally understood that Mtb utilizes host-derived lipids as nutrients to maintain viability during infection. Specifically, various studies have revealed that Mtb can utilize cholesterol and that in animal models Mtb requires cholesterol metabolism to maintain optimal chronic lung infection. Thus, cholesterol utilization by Mtb represents a novel, genetically-validated target for drug discovery.

We previously identified a series of compounds that inhibit cholesterol utilization in Mtb and impair bacterial growth in macrophages. Inhibition by these compounds is dependent on the bacterial adenylyl cyclase (AC) Rv1625c, and the compounds induce 3’,5’-cyclic adenosine monophosphate (cAMP) production in Mtb. From this series, we selected a compound named V-59 to further investigate this mechanism of action and its therapeutic potential. We determined that V-59 selectively activates Rv1625c to increase cAMP synthesis. Unexpectedly, we found that the six-helical transmembrane domain of Rv1625c is necessary for the complete degradation of cholesterol in Mtb, thereby linking the target of V-59 directly to the bacterium’s cholesterol utilization pathway. Additionally, we found that inducing cAMP production via a TetOn system is sufficient to block cholesterol utilization in Mtb, and disrupts this pathway in a dose-dependent manner. V-59 was optimized through medicinal chemistry efforts, resulting in an orally available lead compound (mCLB073) that displays in vivo efficacy in mice chronically infected with Mtb. Collectively, our results reveal that inducing AC activity in Mtb is a previously-unexploited approach that can block cholesterol utilization to inhibit Mtb viability during infection. Moreover, chemically activating bacterial AC enzymes is an unconventional antibacterial target, and this study is the first effort to examine the therapeutic potential of this mechanism of action against a bacterial pathogen.